ELISA的方法檢測多肽與小鼠卵巢癌標(biāo)志物CA125測定試劑盒的結(jié)合情況。準(zhǔn)備將合成多肽(其長度大約為8~10個(gè)氨基酸)包被于ELISA板,再加入小鼠卵巢癌標(biāo)志物CA125測定試劑盒,一抗、酶標(biāo)二抗。請問在包被的過程中應(yīng)采取哪種包被緩沖液?固相載體是否要特殊處理?封閉液用BSA可以嗎?
1、多肽抗原的包被一般需先使其與無關(guān)蛋白質(zhì)如牛血清白蛋白質(zhì)(BSA)等偶聯(lián),借助于偶聯(lián)物與固相載體的吸附,間接地結(jié)合到固相載體表面。
2、包被用抗原或抗體的濃度,包被的溫度和時(shí)間,包被液的pH等應(yīng)根據(jù)試驗(yàn)的特點(diǎn)和材料的性質(zhì)而選定。抗體和蛋白質(zhì)抗原一般采用pH9.6的碳酸鹽緩沖液作為稀釋液,也有用pH7.2的磷酸鹽緩沖液及pH7~8的Tris-HCL緩沖液作為稀釋液的。通常在ELISA板孔中加入包被液后,在4-8℃冰箱中放置過夜,37℃中保溫2小時(shí)被認(rèn)為具有同等的包被效果。
3、zui常用的封閉劑是0.05%-0.5%的牛血清白蛋白,也有用10%的小牛血清或1%明膠作為封閉劑的。脫脂奶粉也是一種良好的封閉劑,其zui大的特點(diǎn)是價(jià)廉,可以高濃度使用(5%)。
顯色終止后出現(xiàn)黑色絮狀沉淀可能為包被板上的蛋白,酶標(biāo)記物,檢測物,封閉液中蛋白變性以及顯色反應(yīng)TMB過量,不溶,沉淀出來。
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